Pyrophosphorolysis activated polymerization (PAP) is a process involving pyrophosphorolysis-mediated activation of pyrophosphorolysis-activatable primers hybridized with target or template nucleic acids followed by extension of the activated primers. Pyrophosphorolysis-activatable primers typically include terminator nucleotides, such as dideoxynucleotides (ddNMPs) at their 3′-termini. These terminator nucleotides must generally be enzymatically removed from such hybridized, blocked primers via pyrophosphorolysis reactions that consume pyrophosphate to produce the activated primers in order for primer extension to proceed. Since primer activation typically requires perfectly matched primers and target nucleic acids, these reactions generally produce limited, if any, non-specific products due, for example, to primer-dimer formation or other false priming artifacts. Exemplary applications of PAP include providing alternative methods for amplifying nucleic acids (e.g., as part of a rare allele detection process, a somatic mutation detection assay, etc.), among other applications.
As referred to above, certain pre-existing PAP-related approaches use ddNMP-terminated primers and efficient ddNMP-incorporating thermostable DNA polymerases (e.g., mutant enzymes that contain an “F to Y” mutation in “Helix O”) to effect pyrophosphorolysis of primers with a 3-terminal ddNMP moiety in the presence of pyrophosphate (PPi) after primer binding/annealing to template. These approaches generally have various disadvantages. For example, these techniques typically use very low, limiting concentrations of dNTPs, which reduces (slows down) the extension rate (number of nucleotides polymerized per second). More specifically, when a 3′-terminal ddNMP is released as a ddNTP in these reactions, the very low concentration of dNTPs (i.e., the “pool size”) increases the likelihood of reincorporating the ever-increasing concentration of ddNTPs (resulting from successive rounds of PAP) prior to full-length primer extension (required for efficient PCR). This reincorporation is generally undesired, requiring additional PAP to remove the incorporated ddNMP and further contributes to incomplete primer extension and compromises the efficiency of full-length primer extension products in each cycle. Moreover, many of these pre-existing PAP approaches are capable of generating only very short amplicons, e.g., in some cases even shorter than primer dimers. Thus, the PCR cycle efficiency of these previous approaches is generally impaired, typically requiring many cycles to detect low abundance targets.
From the foregoing, it is apparent that additional PAP-related methods are desirable. The present invention provides PAP-related methods that utilize 2′-terminator nucleotides, as well as a variety of additional features that will be apparent upon a complete review of the following disclosure.